Lecture 12 Flashcards
Explain how nAChR’s form and what their role is
ACh is secreted by vertebrate pre-synaptic motor neurons which lead to the activation of ion channels called nAChR in post-synaptic muscle cells. nAChR are able to conduct Na which causes depolarization of the cell, leads to an action potential, leads to Ca channels activation/opening, causes an influx of Ca ions, which causes muscle contraction. This is an example of an EPSP.
Discuss the post-synaptic potentials mediated by Cl- channels
Kakuza et al discovered a shift in post-synaptic responses in mouse superior olive neurons to the neurotransmitter glycine.
Glycine activates post-synaptic Cl- channels which are also known as glycine receptors.
A developmental shift in the concentration of Cl out vs Cl in causes GABA and Glycine receptors to change from excitatory to inhibitory
Who is in the Cys-loop family? What are some characteristics of this family?
nACh, Glycine, and GABA are all part of the ligand-gated ion channels known as the cys-loop channels.
These channels are pentameric (meaning that they contain 5 protein subunits to make up a channel)
Each subunit contains an extracellular poor loop bearing 2 cysteine amino acids. The cysteine is present so that it can stabilize the loop, hence the name cys-loop channel.
Cys-loop channels can be anionic (GABA, Glycine for Cl-) or they can be cationic (nACh for Na+ and K+)
What is biochemical isolation? Give an example?
Biochemical isolation is the extraction of desired channel proteins or subunits.
nAChR’s were first isolated from the electric organs of the torpedo ray. The torpedo ray contained modified muscle cells in the electric organs that are packed with cholinergic synapses and nAChR’s. Alpha snake bungarotoxin is a peptide toxin that is known to bind very strongly to nAChR’s. So the used that toxin as bait to fish out all of the nAChR’s from the protein extracts of the electric organs of the torpedo ray. They found that the nAChR’s were composed of 5 proteins: 2 alpha, 1 beta, 1 gamma, and 1 delta.
What is sequencing? Give an example
Sequencing is the next step for studying ion channel structures. In this step the purified channel proteins/subunits are sequenced, the corresponding mRNA’s are found and then those are also sequenced (very laborious research). They found that the 4 nAChR subunits were very similar in that they each contained 4 TMH’s dubbed M1 to M4, a large extracellular N-terminal loop containing 2 cysteines, an extended intracellular loop between M3 and M4 helices, and lastly an extracellular C-terminus.
Digression on ion channel protein sequencing
Ion channel properties arise from the amino acids that are associated with them. Amino acids can be arranged in different sequences to create different secondary structures such as alpha helices, beta sheets, or disordered loops. Amino acids have polar side groups that contribute to either hydrophobicity or hydrophilicity. Lastly, some amino acids have charges, for example glutamic and aspartic acid are negatively charged and histidine, lysine, arginine are positively charged.
What kind of amino acids to TMH’s have?
TMH’s contain mostly hydrophobic amino acids.
Digression on ion channel protein sequencing pt 2
The secondary structures come together and form tertiary structures, this is the final form of the folded protein/subunit. The tertiary structures come together and form the quaternary structure and this is what the ion channel is. nAChR channel is an example of quaternary structure made up of 5 subunits.
Remember the voltage sensor TMH’s on Nav, Kv, and Cav channels?
S4 helices contain hydrophobic amino acids. Positively charged lysine and arginine are crucial for voltage sensing.
Remember the voltage sensor TMH’s on Nav, Kv, and Cav channels?
S2-S3 helices contain hydrophobic amino acids. They also contain negatively charged glutamate and aspartate residues that act as counterions to the S4 charges
More on sequencing (step 2)
Using the deciphered protein sequences, researchers were able to sequence the corresponding mRNA’s. This is a crucial step because it allows you to find out what gene was responsible for coding the ion channel/receptor in question.
Further sequencing and cloning discovered a whole super family of cys-loop channels
(step 2)
GABA receptors, glycine receptors, seratonin receptors, invertebrate MOD-1 receptor, glutamate gated receptor, and EXP-1 (GABA) receptor
GABA Receptor (step 2)
anionic (conducts Cl-), found at inhibitory synapse, vertebrates and invertebrates.
Glycine receptor (step 2)
anionic, inhibitory synapse, (vertebrates/chordates)
Seratonin receptors (step 2)
cationic (Na, Ca), excitatory synapse, vertebrates
